Genomic and immunocyte characterisation of bloodstream infection caused by Klebsiella pneumoniae

Objectives The aim of this study was to evaluate the characteristics of immunocyte associated with bloodstream infection (BSI) caused by Klebsiella pneumoniae (Kpn). Methods Patients with BSI-Kpn were included from 2015 to 2022 in our hospital. Immunocyte subpopulations of enrolled BSI-Kpn patients were tested on the same day of blood culture using multicolor flow cytometry analysis. Antibiotic susceptibility test was determined by agar dilution or broth dilution method. All included isolates were subjected to whole genome sequencing and comparative genomics analysis. Clinical and genetic data were integrated to investigate the risk factors associated with clinical outcome. Results There were 173 patients with non-duplicate BSI-Kpn, including 81 carbapenem-resistant Kpn (CRKP), 30 extended-spectrum β-lactamases producing Kpn (ESBL-Kpn), 62 none CRKP or ESBL-Kpn (S-Kpn). Among 68 ST11-CRKP isolates, ST11-O2v1:KL64 was the most common serotypes cluster (77.9%, 53/68), followed by ST11-OL101: KL47 (13.2%, 9/68). Compared with CSKP group, subpopulations of immunocyte in patients with CRKP were significantly lower (P < 0.01). In patients with ST11-O2v1:KL64 BSI-Kpn, the level of cytotoxic T lymphocytes (CD3 + CD8 +) is the highest, while the B lymphocytes (CD3-CD19 +) was the least. In addition, the level of immunocyte in patients with Kpn co-harbored clpV-ybtQ-qacE were lower than that in patients with Kpn harbored one of clpV, ybtQ or qacE and without these three genes. Furthermore, co-existence of clpV-ybtQ-qacE was independently associated with a higher risk for 30-day mortality. Conclusions The results demonstrate that patients with BSI-CRKP, especially for ST11-O2v1:KL64, exhibit lower leukomonocyte counts. In addition, BSI-Kpn co-harbored clpV-ybtQ-qacE is correlated to higher 30-day mortality. Supplementary Information The online version contains supplementary material available at 10.1186/s12941-024-00721-3.


Introduction
Klebsiella pneumoniae (Kpn) is one of significant pathogens for community-acquired and hospitalacquired infections, especially for bloodstream infection (BSI).In the last decade, the rate of BSI caused by carbapenem-resistant K. pneumoniae (CRKP) is trending upwards from 7.0% in 2014 to 19.6% in 2019 [1].In addition, BSI-CRKP were associated with high mortality due to few therapeutic options [2,3].Recently, several novel antibiotics are available to combat CRKP, however, there have been reports of resistance to novel ß-lactams and ß -lactamase inhibitors [4,5].Therefore, clinical treatment for BSI-Kpn could not just rely upon antibiotics, and the interaction between CRKP and the host immune response needs to be further investigated.
Recent studies demonstrated that Kpn could enhance resistance by mediating cytokine levels and immune cell recruitment in host, resulting in disease-tolerant immune response [6][7][8].In addition, several other evidence demonstrated that Kpn, especially for ST258 CRKP could actively subvert host defence by different immune evasion strategies, such as limiting C3b deposition, triggering apoptosis in macrophages and secretion of several siderophores [6].Notably, ablating host defence by Kpn could cause a more pronounced bacteremia and accelerated mortality in infected mice [9,10].However, the majority of evidence on the interplay between Kpn and host immune response were obtained by infecting rodents, chiefly by mice [8][9][10].Few studies addressed the response of immune cells to counteracting BSI-Kpn, especially for ST11 CRKP.Therefore, this present study was conducted to elucidate the immune cells response against different BSI-Kpn in clinic.In addition, clinical and genetic data were integrated to further investigate the risk factors associated with clinical outcome.

Patients and clinical data
A retrospective study was conducted to obtain profiles of the immune response in patients with BSI-Kpn and identify the risk factors associated with 30-day mortality.Clinical diagnosis of BSI-Kpn was screened by positive blood culture at The First Affiliated Hospital, Zhejiang University School of Medicine from 2015 to 2022.Meanwhile, patients tested immunocyte subpopulations on the same day of blood culture.The medical records of included patients were reviewed.The detailed analysis data and definitions were obtained as described in our previous study [11,12].The 30-day mortality was used as treatment outcomes.This study was approved by the recommendations of the Ethics Committee of The First Affiliated Hospital, Zhejiang University School of Medicine.

Strain collection and antibiotic susceptibility test
A total of 173 BSI-Kpn strains were collected in the study.The minimal inhibitory concentration (MIC) of 20 antibiotics against BSI-Kp isolates were determined by agar dilution method, while antimicrobial sensitivity of tigecycline and polymyxin B was done by broth dilution method.
Extended-spectrum-β-lactamase (ESBL)-producing isolates were confirmed for ESBLs production by disk synergy test [15].CRKP was defined as Kpn strain resistant to imipenem or meropenem or ertapenem.Non-CRKP or non-ESBL-Kpn was considered as "susceptible" Kpn (S-Kpn).ESBL-Kpn and S-Kpn belong to carbapenem-sensitive K. pneumoniae (CSKP).The details of antibiotics were consistent with our previous study [16].The results for test antibiotics were interpreted according to Clinical and Laboratory Standards Institute (CLSI) guidelines [15].
All sequence data has been deposited under BioProject accession No. PRJNA778807 and PRJNA1056658.

Statistical analysis
The results for abnormal distribution of continuous variables were presented as median (interquartile range) and analyzed using chi-square test.Multivariate analysis was performed after identifying variables with a P-value of < 0.05 in the univariate analysis.A two-tailed P value < 0.05 was considered to be statistically significant.GraphPad Prism software (V8.0) and SPSS 23.0 for Windows (SPSS Inc., Chicago, IL, USA) were used to analyze data.The 30-day mortality was established basing on the regression model by employing the R v4.1.2package.

Clinical characteristics in patients with BSI-Kpn
There were 173 patients with non-duplicate BSI-Kpn, including 81 CRKP, 30 ESBL, 62 S-Kpn, were included in this study.The clinical characteristics of all patients are shown in Table 1.There were no significant differences in gender and age.Organ transplant patients were more likely to be infected with CPKP.In addition, CRKP patients also underwent more invasive procedures.The levels of albumin and procalcitonin in patients with CRKP were lower than that in patients with CSKP.More patients in CRKP group received aminoglycoside, fosfomycin, tigecycline, polymyxin B, and ceftazidimeavibactam. APACHE-II score and SOFA score in CRKP were higher than that in CSKP (P < 0.05).Furthermore, the 30-day mortality in CRKP was significantly higher than that in CSKP (P < 0.000).

Risk factors for 30-day mortality in enrolled patients
A total of 42 BSI-Kpn patients died, including 12 CSKP and 30 CRKP.The results of univariate analysis and logistic regression was shown in Table 3 and Supplementary Table 5.Compared to the survival group, BSI-Kpn patients with resistance genes (bla KPC- like , bla TEM-1B , rmtB, dfrA-like, aadA, ramR) and virulence genes (AAA92657, clpV, ybtQ, ybtU) caused higher mortality (P < 0.05).Notably, BSI-Kpn isolates co-harbored clpV, ybtQ and qacE resulted in higher 30-day mortality than isolates carried one of three or none of them (Fig. 4).Lower count of white blood cell, hemoglobin, platelet, and cholinesterase showed significant associations with 30-day mortality in a univariate analysis.In addition, use of glucocorticoid and immunosuppressant before BSI-Kpn are risk factors for 30-day mortality.Patients with longer ICU stay and more invasive procedures, such as mechanical ventilation, hemodialysis, urinary catheterization, arterial catheterization and stomach tube, had a higher mortality rate (P < 0.05).Tigecycline and combination therapy after diagnosis led to increased 30-day mortality.Further logistic regression established multivariate predictive model, demonstrating length of ICU stay, hemodialysis, urinary catheterization, platelet count, APACHE-II score and co-existence of clpV-ybtQ-qacE independently associated with a higher risk for 30-day mortality.

Discussion
Although novel antibiotics have shown promising efficacy against multidrug-resistant bacterial infections, the mortality rate in BSI-CRKP remains high [2,3].Therefore, relying on antibiotics is not enough.Clearance of Kpn requires a complex, multi-faceted response of innate immune cells and ultimately resolved by adaptive immune cells [6,21].Therefore, the role of host immune responses and the interaction between BSI-Kpn and immunocyte in treating deadly infections deserves attention.
Kpn display a population structure characterized by genetic diversity, however, highly successful clonal genetic lineages were observed in CRKP, such as ST258 and ST11 [22,23].However, a previously prevalent subclone OL101:KL47 was replaced by O2v1:KL64 in the predominant clone ST11 [23].In the present study, we also found ST11-O2v1:KL64 was the most common serotypes cluster in BSI-CRKP, which clustered with one sublineage of ST11-OL101: KL47 as well.The abundant genetic diversity between CRKP and CSKP may result in heterogeneity of interaction with host immune responses [24].
Several studies demonstrated that Kpn, especially for CRKP, could frustrate normal immune responses, capitalize upon states of immunocompromise, and cause infections with high mortality [21,25].Our results demonstrated the absolute numbers of tested immunocyte in patients with CRKP were significantly lower than that in CSKP (P < 0.01).Previous study found Kpn stimulated highly purified human blood B cells [26].Our study further demonstrated the B lymphocytes (CD3-CD19 +) in ST11-O2v1:KL64 showed the lowest level.Therefore, genetic diversity of BSI-Kpn impacts the outcomes of the interactions between the Kpn and each of these host immunocyte responses.Previous studies revealed bacterial factors of hypervirulent Kpn, such as capsule polysaccharides, lipopolysaccharide, type VI secretion system (T6SS) resulted in a common evasion strategy based on phagocytosis evasion, while CRKP showed heterogeneous evasion strategies, leading to prolong intracellular survival [7,9,27].However, few studies have addressed the relationship between genetic diversity of Kpn and inflammatory response.To obtain comprehensive information, it will be necessary to assess the mechanism of common and unique interaction between BSI-Kpn heterogeneity and immune cells.
Although some studies have demonstrated CRKP infections were associated with BSI mortality in several clinical series, the risk factors remain inconsistent [2,3,28].CD4 + /CD8 + T lymphocytes ratio < 1 was identified as independent risk factors for ICU admission patients with CRKP infections [28].Similarly, we found the level of CD8 + in ST11-O2v1:KL64 among patients with CRKP was higher than that in other serotypes and STs combinations.The level of immunocyte in isolates co-harbored clpV-ybtQ-qacE were lower than that in isolates harbored one of clpV, ybtQ or qacE and without these three genes.Furthermore, co-existence of clpV-ybtQ-qacE was independently associated with a higher risk for 30-day mortality.Previous studies reported T6SS core component (clpV) used by Salmonella to survive within host macrophages [29].ybtQ, encoding a transporter for the siderophore yersiniabactin, played an important role for the ability of Kpn to maintain infection in a mammalian host [30].qacE, as disinfectant resistance gene, reduced biocide susceptibility [31].Taken together, these data show correlation among virulence, antibiotic resistance and inflammatory response, which could affect the ability to clear Kpn and increase the risk of death.
This study firstly provides an insight into interactions between different phylogenetic BSI-Kpn and immunocyte subpopulations in clinic.However, there were also several limitations in the present study.First, the immune cells were not dynamically observed during the infections.Therefore, it is unable to fully elucidate the changes in host inflammatory response throughout the entire BSI process.In addition, the number of isolates in this study remains not enough.Above all, there is a pressing need to focus on the dynamics and outcomes of the interactions between BSI-Kpn heterogeneity and host immune response.Fig. 4 Kaplan-Meier survival estimates for patients with BSI-Kpn co-harbored clpV-ybtQ-qacE, harbored one of clpV, ybtQ or qacE, and without these three genes.A significant difference was found in the 30-day mortality between the 3 groups (P < 0.0001)

Table 1
Clinical characteristics of all patients with BSI-Kpn

Table 3
Logistic regression model of isk factors for 30-day mortality